The overall goal of this proposal is to construct a vaccine that is broadly protective against multiple enteropathogens of public health importance to the U.S. population. These enteropathogens include Shigella (including S. dysenteriae 1, a bioterror threat agent), enterotoxigenic E. coli (ETEC) (the major cause of traveler's diarrhea) and the emerging pathogens shiga toxin producing E. coli (STEC) (main cause of hemolytic uremic syndrome, HUS) and enteroaggregative E. coli (EAEC) (recently recognized as an important agent of pediatric diarrheal disease in the U.S.A). These enteropathogens are all Category B risk agents of biodefense concern. A vaccine with broad coverage against this group would be beneficial to multiple segments of the US population, including: 1) adult and child travelers who visit less developed countries where these infections are hyperendemic; 2) children in certain areas high risk areas of the US; 3) and for mass immunization in the face of deliberate bioterror spread most of these pathogens. Immunization by the mucosal route is an effective method for induction of mucosal and systemic immune responses, believed to be important in protection against these enteropathogens. Studies during the first MARGE funding cycle revealed the ability of our live attenuated S. dysenteriae 1 vaccine strain expressing Stx1 B to induce high titer Stx1 neutralizing antibodies in an animal model, in addition to S. dysenteriae 1 LPS specific antibodies that are critical for protection against S dysenteriae 1 disease. The elicitation of antitoxin that neutralizes Stx1 suggests the potential to confer protection against other Shiga toxin expressing pathogens such as STEC. In this application we propose to extend the spectrum of coverage of our multivalent live vector enteric vaccine by expressing protective antigens from STEC and EAEC. In a rational step-wise manner, Shiga toxin B subunits from Stx1 and Stx2, a chimeric toxoid Stx2A1B, and the protective AAF/II fimbrial antigen from EAEC will be engineered in attenuated derivatives of S. dysenteriae 1, and S. flexneri serotypes 3a and 6 using a novel ssb-stabilized plasmid system, Optimal live vector-antigen configurations will be evaluated for protection against STEC infection in a rabbit model or inhibition of cell binding and biofilm formation by EAEC. Combined with ongoing Shigella-ETEC vaccine constructions, a tetravalent enteropathogen vaccine will be developed.